This invention relates to methods of detecting hybridization between nucleic acids or protein/nucleic acid interactions. In particular this invention pertains to nucleic acid probes containing a fluorescent nucleotide analogue whose fluorescence level increases when the probe hybridizes to a target nucleic acid or when the probe is bound by a protein.
One of the most specific molecular recognition events takes place when a strand of nucleic acid anneals (hybridizes) to its complement. A single-stranded oligonucleotide probe can find a complementary strand in the presence of a large excess of other nucleic acids. This process has facilitated the exploration of gene structure and organization, the measurement of gene expression and function, and the detection and characterization of a wide variety of pathologies.
Hybridization based assays typically require detection and/or quantification of a hybrid duplex formed between a probe nucleic acid and its corresponding target nucleic acid. However, because the measurable changes in the physical properties of nucleic acids that occur upon hybridization are rather small, such assays most frequently utilize labels attached either to the nucleic acid probes or to the target nucleic acids to detect the hybrid duplexes. After hybridization, typically the hybridized nucleic acids are immobilized (e.g., attached to a membrane) and the unhybridized nucleic acids are washed away. The immobilized label is then detected and/or quantitated to provide a measure of the hybrid duplex.
The requirement that unhybridized probes be separated from hybridized probes precludes the use of hybridization for real-time monitoring of nucleic acid syntheses or protein-nucleic acid interactions or for location specific nucleic acids in living cells. In addition, the need to immobilize hybrids on a solid surface limits sensitivity, since probes bind non-specifically to surfaces. While several schemes have been put forward for detecting specific nucleic acids in homogeneous solutions (see, e.g., Heller et al. European Patent Application 82303699.1, Morrison et al. (1989) Anal. Biochem., 183: 231-244, Cardullo et al. (1988) Proc. Natl. Acad. Sci. USA, 85: 8790-8794, Morrison et al. (1993) Biochem., 32: 3095-3104, and Sixou et al. (1994) Nucl. Acids Res., 22: 662-668), these methods are typically unsuitable for real-time measurements or use in living cells.
Newer approaches for the detection of nucleic acid hybridizations and protein-nucleic acid interactions typically rely on energy transfer between a fluorophore and a quencher molecule or a second fluorophore (e.g., a fluorescence resonance energy transfer system). Thus, for example, a lumazine derivative has been used in conjunction with a bathophenanthroline-ruthenium complex as an energy transfer system in which the lumazine derivative acted as an energy donor and the ruthenium complex acted as an energy receptor. The lumazine derivative and ruthenium complex were attached to different nucleic acids. Energy transfer occurred when the two compounds were brought into proximity resulting in fluorescence. The system provided a mechanism for studying the interaction of molecules bearing the two groups (see, e.g., Bannwarth et al., Helvetica Chimica Acta. (1991) 74: 1991-1999, Bannwarth et al. (1991), Helvetica Chimica Acta. 74: 2000-2007, and Bannwarth et al., European Patent Application No. 0439036A2).
Another approach utilizes nucleic acid probes bearing a fluorophore and a quencher molecule. The probes were self-complementary and adopted a hairpin conformation in solution. The hairpin juxtaposed the fluorophore and the quencher thereby reducing or eliminating fluorescence of the fluorophore. When the probes hybridized to a target nucleic acid, they linearized, separating the fluorophore from the quencher molecule and thereby providing a fluorescent signal (see Tyagi and Kramer et al. (1996) Nature Biotechnology, 14: 303-308).
Both of these approaches required a fluorescent compound and a second fluorophore or a quencher. Most fluorescent compounds, however, generally suffer the disadvantage that the fluorescent complexes and their binding moieties are relatively large. The presence of large fluorescent labels and associated linkers may alter the mobility of the nucleic acid, either through a gel as in sequencing, or through various compartments of a cell.
In addition, the presence of these markers alters the interaction of the labeled nucleic acid with other molecules either through chemical interactions or through steric hindrance. The presence of these markers thus makes it difficult to study the interactions of DNA with other molecules such as other nucleic acids or proteins.
This invention provides new methods and compositions for the detection of nucleic acid interactions with other nucleic acids or with proteins. The methods and compositions utilize fluorescent nucleotide analogs as fluorescent moieties and thus do not suffer from the limitations described above.
The methods of this invention generally utilize a nucleic acid (e.g., an oligonucleotide) that contains one or more fluorescent nucleotide analogues. The fluorescence of the nucleotide analogues is quenched (reduced) when they are incorporated into the oligonucleotide (see, e.g., U.S. Pat. No. 5,525,711 and Hawkins et al. (1995) Nucl. Acids Res., 23: 2872-2880. However, when the fluorescent nucleotide analogue is removed from the quenching influence of neighboring bases (e.g, present in a loop) fluorescence activity is partially or completely restored. Without being bound by a particular theory, it is believed that alteration of the normal conformation (e.g., base stacking) of the oligonucleotide at the location of the fluorescent nucleotide analogue reduces and/or eliminates the quench thereby causing an increase in fluorescence.
Thus, in one embodiment, this invention provides methods of detecting the presence, absence, or quantity of a target nucleic acid. The methods involve contacting the target nucleic acid with a nucleic acid probe where the nucleic acid probe comprises a fluorescent nucleotide located in the probe such that, when the probe hybridizes to the target nucleic acid, the fluorescent nucleotide is present in a loop that does not participate in complementary base pairing with a nucleotide of the target nucleic acid; and detecting the fluorescence produced by the fluorescent nucleotide, when said probe forms a hybrid duplex with said target nucleic acid. In one preferred embodiment, the loop ranges in length from about 1 to about 100 nucleotides when the probe hybridizes to said target nucleic acid. In particularly preferred probes, the loop is an insertion in said nucleic acid probe which is otherwise complementary to said target nucleic acid or to a contiguous subsequence of said target nucleic acid. In some preferred embodiments, the insertion is three nucleotides in length and comprises two nucleotides each adjacent to the fluorescent nucleotide. In particularly preferred embodiments, at least one nucleotide adjacent to the fluorescent nucleotide is a purine (e.g., adenosine), and in still more preferred embodiments, the fluorescent nucleotide is bordered by at least two adjacent purines (e.g., adenosine) in both the 5xe2x80x2 and 3xe2x80x2 direction. In a most preferred embodiment, the insertion is a single base insertion; the fluorescent nucleotide.
In yet another embodiment, the insertion is self-complementary and forms a hairpin in which the fluorescent nucleotide is present in the loop of said hairpin and does not participate in complementary base pairing. The nucleotides comprising the loop can be selected such that they are not complementary to the corresponding nucleotides of the target nucleic acid when said probe is hybridized to said target nucleic acid and where said probe is complementary to at least two non-contiguous subsequences of said target nucleic acid.
In another embodiment, the fluorescent nucleotide is present in a terminal subsequence of the nucleic acid probe where the terminal subsequence does not hybridize to the target nucleic acid when the remainder of said nucleic acid probe hybridizes to said target nucleic acid. The terminal subsequence may form a terminal hairpin by hybridization with a second subsequence of the probe such that the fluorescent nucleotide is present in a loop of said hairpin and does not participate in complementary base pairing.
Particularly preferred detection methods involve detecting an increase in fluorescence of the fluorescent nucleotide when the probe forms a hybrid duplex with the target nucleic acid. In preferred label oligonucleotides, the fluorescent nucleotides include one or more of any of the fluorescent nucleotide analogues described herein.
In another embodiment, this invention provides methods of amplifying nucleic acids. The methods involve providing in a nucleic acid amplification mixture a label oligonucleotide comprising a fluorescent nucleotide analog, where the label oligonucleotide comprises a nucleotide sequence such that when an amplification product is formed in a nucleic acid amplification reaction using the mixture, the label oligonucleotide hybridizes to the amplification product or to a subsequence thereof and forms a loop in which said fluorescent nucleotide analogue does not participate in complementary base pairing. The hybridization of the label oligonucleotide to the amplification product produces a change in fluorescence that is detected. The label oligonucleotide can be a primer in the amplification reaction or it can be a separate label that does not act as a primer in the amplification reaction. The label oligonucleotide can include any of the above-described probes or label oligonucleotides described herein.
In still yet another embodiment, this invention provides fluorescent labels. The labels comprising any of the probes or label oligonucleotides described herein hybridized to a target nucleic acid forming a hybrid duplex in which said fluorescent nucleotide does not participate in complementary base pairing with a nucleotide of said target nucleic acid.
It was also a discovery of this invention that the signal produced by a label oligonucleotide hybridizing in a complex mixture of nucleic acids can be improved by cutting (e.g., by shearing, acid hydrolyzing, or restriction digesting) the other nucleic acids (e.g, target, template, or other nucleic acids present in a sample) to a characteristic length and/or by providing an overabundance of the label oligonucleotide. This cutting step can be practiced in any of the methods described herein. The cutting alters hybridization kinetics to favor binding by the label oligonucleotide. While cutting to any shorter length improves label oligonucleotide binding so long as the target nucleic acid is not destroyed, in a preferred embodiment, the nucleic acids are cut to a length that substantially approximates the length of the target nucleic acid(s) (e.g., to a length no greater than 20 times, preferably no greater than 10 times, more preferably to a length no greater than 5 times, and most preferably to a length no greater than 2 times, 1.5 times or even no greater than 1 times the length of the label oligonucleotide). In a most preferred embodiment, the characteristic length is approximately the length of the label oligonucleotide.
This invention also provides nucleic acid amplification mixtures comprising a label oligonucleotide and a DNA polymerase, wherein said label oligonucleotide comprises a fluorescent nucleotide analog and has a nucleotide sequence such that when an amplification product is formed in a nucleic acid amplification reaction using the amplification mixture, the label oligonucleotide hybridizes to the amplification product or to a subsequence thereof and forms a loop in which said fluorescent nucleotide analogue does not participate in complementary base pairing. Again, the label oligonucleotide can be any of the probes or label oligonucleotides described herein.
This invention also provides kits for performing nucleic acid amplifications or for detecting the presence absence or quantity of a nucleic acid in a sample. The kits comprise a container containing any of the probes or label oligonucleotides described herein. The kit can further comprise, one or more restriction enzymes, a buffer, and/or any of the other reagents useful for practicing the method to which the kit is directed.
The terms xe2x80x9cnucleotidexe2x80x9d or xe2x80x9cnucleotide monomerxe2x80x9d, as used herein, refer to the xe2x80x9cstandardxe2x80x9d nucleotides; adenosine, guanosine, cytidine, thymidine, and uracil, or derivatives of these nucleotides. Such derivatives include, but are not limited to, inosine, 5-bromodeoxycytidine, 5-bromo-deoxyuridine, N6-methyl-deoxyadenosine and 5-methyl-deoxycytidine. The terms also include nucleotide analogues, more preferably fluorescent nucleotide analogues including, but not limited to 2-amino purine, any of the pteridine nucleotides disclosed herein, or any of the other fluorescent nucleotides disclosed herein.
A xe2x80x9cfluorescent nucleotidexe2x80x9d or a xe2x80x9cfluorescent nucleotide analoguexe2x80x9d refers to a nucleotide or nucleotide analogue that is capable of emitting a fluorescent signal when illuminated with light of an appropriate wavelength. The fluorescent signal is reduced or eliminated when the nucleotide is incorporated into an oligonucleotide. However, as long as the nucleotide analogue emits a fluorescent signal with a quantum yield above 0.04, more preferably above 0.1 and most preferably above 0.15 when it exists as a monomer in an aqueous solution it is regarded as a fluorescent nucleotide.
A xe2x80x9cpteridine nucleotidexe2x80x9d or a xe2x80x9clumazine nucleotidexe2x80x9d refer to fluorescent nucleotide analogues in which the base portion of the molecule is a pteridine/pteridine derivative or a lumazine/lumazine derivative respectively. It is recognized that lumazines are a subclass of pteridines.
The term xe2x80x9cfluorescence intensityxe2x80x9d refers to the quantum yield of a molecule. Quantum yield is typically expressed as a relative quantum yield (relative to a standard) and is given as:   Q  =                    Q        std            (                        ∫                      Em            std                                    Abs          std                    )                      ∫                  Em          Sample                            Abs        Sample            
where Qstd is the quantum yield of a standard EMstd is the emission of a standard, ABSstd is the absorbance of a standard EMsample is the emission of a sample, and ABSsample is the absorbance of a standard. In a preferred embodiment, the standard is quinine sulfate (QS) which has a quantum yield of 0.51 as reported by the National Bureau of Standards (Velapoldi and Mienenz (1980) Nat. Bur. Standards Vol. 260-64). Thus, in a preferred embodiment, the relative quantum yield is thus the ratio of the integral of the emission scan of quinine sulfate (Emstd=EMQS) divided by the absorbance (optical density) of the quinine sulfate at the excitation wavelength (Absstd) to the integral of the sample emission scan (EmSample) divided by the absorbance (optical density) of the sample at the excitation wavelength. Relative quantum yield thus provides a measure of the efficiency of the fluorophore in converting absorbed light to emitted light. Methods of determining relative quantum yield are well known to those of skill in the art (see, e.g., Velapoldi and Mienenz, supra.) Fluorescence intensity is measured by any of a number of means well known to those of skill in the art. In a preferred embodiment, fluorescence intensity is determined in an aqueous solution using a fluorometer.
The term xe2x80x9coligonucleotidexe2x80x9d, as used herein, refers to a molecule comprised of two or more deoxyribonucleotides, ribonucleotides, modified ribonucleotides, modified dexoyribonucleotides, fluorescent or non-fluorescent ribonucleotide analogs, or fluorescent or non-fluorescent deoxyribonucleotide analogs. The exact size of an oligonucleotide depends on many factors and the ultimate function or use of the oligonucleotide. Generally, chemically synthesized oligonucleotides range is length from 2 to 200 bases, although, it is well known that oligonucleotides may be ligated together to provide longer sequences. As used herein, the term xe2x80x9coligonucleotidexe2x80x9d also encompasses these longer sequences. It is also recognized that double-stranded polynucleotides may be created by hybridization with a complementary sequence or enzymatically through primer extension.
The term xe2x80x9clabel oligonucleotidexe2x80x9d, as used herein, refers to an oligonucleotide incorporating one or more fluorescent nucleotide analogues. The fluorescence activity of the nucleotide analogue(s) may be quenched partially or to a non-detectable level when the label oligonucleotide achieves a substantially linear conformation (i.e., the constituent bases, more particularly the fluorescent nucleotide(s), participate in normal base stacking). Preferred label oligonucleotides of this invention are capable of achieving a conformation, when hybridized to themselves or another nucleic acid or when bound by a nucleic acid binding protein, in which the quench (reduction of fluorescence activity (intensity) of the fluorescent nucleotide(s)) is diminished or eliminated resulting in a label oligonucleotide having increased fluorescence when present in that conformation. The label oligonucleotides of this invention are distinguished from labeled oligonucleotides which are oligonucleotides to which is attached a label. The labeled oligonucleotide can of course be attached to (labeled with) a label oligonucleotide of the present invention either directly through a phosphodiester linkage or indirectly through a linker.
xe2x80x9cSubsequencexe2x80x9d refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.
The terms xe2x80x9ctarget nucleic acidxe2x80x9d or xe2x80x9ctarget oligonucleotidexe2x80x9d refer to the nucleic acid sequence or nucleic acid subsequence that is to be detected using one or more label oligonucleotides of this invention. The label oligonucleotides typically hybridize to all or a part of the target nucleic acid under stringent conditions.
The term xe2x80x9ccorresponding nucleotidexe2x80x9d, is used to refer the position of a nucleotide in a first nucleic acid by reference to a second nucleic acid. Thus, a corresponding nucleotide refers to a nucleotide that would form a complementary base pair (i.e. would hydrogen bond) with the nucleotide in the first nucleic acid to which the correspondence is drawn, were the first and second nucleic acids perfectly complementary and hybridized under stringent conditions.
Hybridization refers to the specific binding of two nucleic acids through complementary base pairing. Hybridization typically involves the formation of hydrogen bonds between nucleotides in one nucleic acid and their corresponding nucleotides in the second nucleic acid.
xe2x80x9cBind(s) substantiallyxe2x80x9d refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
The phrase xe2x80x9chybridizing specifically toxe2x80x9d, refers to the binding, duplexing, or hybridizing of a molecule only to a particularly nucleotide sequence or subsequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA). The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
The term complementary base pair refers to a pair of bases (nucleotides) each in a separate nucleic acid in which each base of the pair is hydrogen bonded to the other. A xe2x80x9cclassicalxe2x80x9d (Watson-Crick) base pair always contains one purine and one pyrimidine; adenine pairs specifically with thymine (A-T), guanine with cytosine (G-C), uracil with adenine (U-A). The two bases in a classical base pair are said to be complementary to each other.
A xe2x80x9cnucleic acid amplification mixturexe2x80x9d refers to the reaction mixture used to amplify a nucleic acid. The amplification may be by any method including but not limited to PCR, long range PCR, ligase chain reaction, self-sustained sequence replication, and the like. Typical nucleic acid amplification mixtures (e.g., PCR reaction mixture) include a nucleic acid template that is to be amplified, a nucleic acid polymerase, nucleic acid primer sequence(s), and nucleotide triphosphates, and a buffer containing all of the ion species required for the amplification reaction.